The endogenous cannabinoids and their attending G protein-coupled receptors are components of a neuromodulatory system that regulates multiple brain functions, including pain and reward. The endocannabinoid anandamide is released from neurons upon demand and undergoes a rapid deactivation process that is thought to involve two sequential steps: carrier-mediated transport into cells and intracellular hydrolysis by fatty acid amide hydrolase-1 (FAAH-1) and FAAH-2. The main objective of the present application is to address two key aspects of anandamide deactivation, which remain incompletely understood: (i) the mechanism and significance of anandamide transport; and (ii) the roles of FAAH-mediated anandamide hydrolysis outside the central nervous system (CNS). In previous studies, funded by this grant, we developed the first brain-impermeant FAAH inhibitor and showed that this O-aryl carbamate derivative (termed URB937) suppresses acute and chronic pain-related responses in rodents by enhancing the intrinsic activity of anandamide at CB1-type cannabinoid receptors located outside the CNS. Furthermore, we molecularly cloned a catalytically defective variant of FAAH-1 (termed FAAH-Like Anandamide Transporter, FLAT) that lacks amidase activity, but selectively binds to anandamide and facilitates the transport of this compound into cells. Finally, we discovered a ligand (ARN272) that inhibits anandamide binding to FLAT, blocks anandamide transport in vitro and interrupts anandamide deactivation in vivo. The present proposal has two primary aims. Aim 1: To define pharmacophore profiles for peripheral FAAH inhibition and identify new brain-impermeant FAAH inhibitors. We will synthesize analogs of URB937 to (i) determine structure-activity relationships for peripheral segregation within the O-aryl carbamate chemotype; and (ii) identify new brain-impermeant FAAH inhibitors, which may be utilized both to investigate the functions of peripheral FAAH and as prototypes for analgesic agents devoid of central side effects. Aim 2: To characterize the role of FLAT in neuronal anandamide transport and discover potent and selective FLAT inhibitors. We will perform three sets of studies: (a) we will conduct computational and mutational analyses aimed at exploring the molecular mechanism(s) through which substrates (such as anandamide) and inhibitors (such as ARN272) interact with FLAT; (b) we will investigate the mechanism through which FLAT mediates anandamide transport; and (c) we will use the scaffold of ARN272 to create novel FLAT ligands that may serve as tools to investigate the functions of FLAT in anandamide transport. These studies are likely to generate new molecular tools that will help elucidate the mechanism of anandamide deactivation and may lead to the discovery of new drug candidates for pain, substance abuse and other human diseases.